The insulin receptor is encoded by a single copy gene located on chromosome 19 in the human. In addition, at least two other highly homologous genes have been identified. One gene encodes the type 1 insulin-like growth factor (IGF) receptor. The third gene in the family encodes an orphan receptor tyrosine kinase that has been named the insulin receptor-related receptor (IRR). We have cloned the cDNA molecules encoding human and mouse IRR. Generally, the predicted structure of IRR is similar to those of the receptors for insulin and IGF-1. However, the phosphotyrosine content of IRR is not increased by incubating the cells with insulin, IGF-1, or proinsulin. Nevertheless, because IRR may be co-expressed in the same cells as the homologous receptors for insulin and insulin-like growth factor-I (IGF-1), we inquired whether IRR might be activated via formation of hybrids with insulin receptors. To test this hypothesis, we co-expressed the human IRR and the human insulin receptor (IR) in NIH-3T3 cells, and observed that a small proportion of each receptor was assembled into IRR/IR hybrids. While insulin was capable of stimulating insulin receptor autophosphorylation in these cells, there was no detectable increase in the total phosphotyrosine content of IRR. Thus, we conclude that IRR/IR hybrids do not play a major role in IRR signal transduction in response to insulin. These observations are consistent with the hypothesis that there is a physiological ligand that binds to the extracellular domain of IRR, thereby specifically regulating its tyrosine kinase activity. To pursue this hypothesis, we used an assay based upon the ability to stimulate autophosphorylation of IRR, and have accomplished 100-fold purification of a protein factor from rat brain that is a candidate to be the ligand for the IRR.